PROJECT SUMMARY Cystic fibrosis (CF) is a debilitating, progressive lung disease that currently has no cure. With proper management, patients can achieve good symptom control and quality of life. Exacerbations are acute episodes of worsening of respiratory symptoms, often triggered by an infection. During exacerbations, excess neutrophil elastase (NE) is released into the lung and causes irreversible damage to lung tissue. This enzyme can be detected in sputum and therefore serves as a biomarker for acute exacerbations. We hypothesize that daily, remote monitoring of this enzyme in CF patient sputum would result in more effective treatment and a reduction in lung tissue damage. Therefore, the objective is to develop a portable, point-of-care assay that can be used by CF patients to monitor NE activity in their sputum. The testing platform will consist of a magnetic chip reader and a disposable biosensor chip that has been pre- functionalized with NE-specific cleavable peptides attached to magnetic nanoparticles (MNPs). The peptides will serve as linkers between the sensor surface and the MNPs. Cleavage of peptides by NE will release the MNP resulting in a quantitative decrease in signal over time. The rate of peptide cleavage correlates with NE abundance. Therefore, when NE increases in sputum, this device will notify clinicians that an acute exacerbation is beginning. The ability to detect and quantify NE activity will be determined by how well this enzyme can cleave surface immobilized peptide substrates. Therefore, in Aim 1, we will use 96-well plate assays to evaluate different surface chemistries for optimal attachment of the peptide. To facilitate efficient cleavage by NE, we will optimize the density and length of the peptide substrate. Once optimized, the peptide substrate will be immobilized to the magnetic sensor surface and the fluorescent reporter used in the 96-well plates will be replaced with MNPs. In Aim 2, addition of either purified NE or patient sputum samples will result in cleavage of the peptide and a decrease in magnetic resistance as the MNPs are released from the sensor surface. This wash-free protease assay can be monitored in real-time enabling a ?sample-to-answer? testing procedure that can be run by untrained professionals. While our proposed research will focus on sputum from CF patients, this assay will be suitable for use by patients with other chromic airway diseases, including Chronic Obstructive Pulmonary Disease (COPD).